Of the species of ants present in the United States, Solenopsis richteri and Solenopsis invicta, commonly referred to as the imported black fire ant and imported red fire ant, respectively, are not indigenous to North America. Both of these species are indigenous to the riverbanks of the Paraguay and Guapore regions of South America. S. geminata is a fire ant that is indigenous to North America.
The imported red fire ant (Solenopsis invicta), a major pest in the Southern United States, has had a large medical and economic impact because of its effects on man, livestock, and agriculture in general. For example, a study at the University of Texas, on a 70-acre tract of land, showed that as S. invicta moved across the effect was on the whole insect fauna. The number of other ant species declined by 70%, while a number of arthropod species, such as spiders, ticks, and other insects, dropped by 40%. It has been noted that fire ants are able to damage the roots of various crops, girdle the stems of young citrus trees, as well as feed on the fruits and flowers of several plants, particularly okra.
The most noticeable concern imposed by S. invicta is its physical and medical impact on man. Fire ants are a major nuisance to humans and other animals due to their powerful, painful sting, from which the name was acquired. In S. invicta, 95% of the venom consists primarily of alkyloids (2,6-disubstituted piperidines). Allergic reactions which can lead to death from anaphylactic shock occur in 1-2% of the population. A 1989 survey announced that 32 persons died from fire ant stings, most of which involved less than five stings. Due to the prevalence of S. invicta, man cannot help but encounter them in any outdoor activity, resulting in between 67,000-85,000 people per year seeking medical treatment for fire ant stings.
In 1957, when it became apparent that fire ants posed a serious problem, the federal government launched a massive campaign to eliminate this pest. In five years, the United States government spent 70 million dollars dispensing chlordane, dieldrin and heptachlor over large areas. While these three poisons did kill fire ants, they also had adverse effects to a variety of other organisms, including the native ant fauna. Also, due to their stability, these compounds were soon detected in the environment and were found to be responsible for the death of birds, fish and reptiles. In view of this, the use of these chemicals immediately ceased. A few years later in the mid-sixties, the compound mirex was developed. It proved effective against S. invicta, but unfortunately, it was also found to be capable of killing all species of ants. Nonetheless, since mirex appeared to have minimal environmental effects and eradication seemed economically feasible, this poison was broadly employed to control fire ant infestation. However, after several years of use, low levels of residues were detected in some non-target organisms. Perhaps more significantly, remnants of this poison were found in adipose tissue from humans and studies suggested that mirex might be a carcinogen. Therefore, in 1978, the Environmental Protection Agency revoked the use of mirex, thus, ending the widespread distribution of this poison. Since that decision there has never been a more intensive effort to control an unwanted pest in the United States.
When the above poisons were used to fight the fire ant infestation, the indigenous species of ants, which happen to be the best defense against the fire ant, were also killed. By eliminating the fire ant""s competition, in combination with their aggressiveness and high rate of reproduction, man played to the strength of S. invicta by clearing the way for it to invade the North American habitat.
Currently, the most common methods of treating fire ant infestation include the use of toxicants (amidohydrozones), juvenile hormone analogs, or avermectin B1a. However, in order to eliminate the colony completely it is necessary to kill the queen, and with the emergence of polygyne (multiple queen) colonies this has become more difficult. Juvenile hormone analogs interfere with the development of the brood, but the entire colony must be reached or it will quickly rebound. It has been observed that sometimes the colony will relocate in response to physical disturbance or stress if members of the colony suddenly die. Therefore a subtler and less invasive means of control would be useful.
In the course of maturation, the fire ant undergoes eight stages of development: eggxe2x80x941st, 2nd, 3rd and 4th instar larvaexe2x80x94prepupaexe2x80x94pupaexe2x80x94adult. The 4th instar larvae plays an important role in the survival of the colony in that it is totally responsible for the digestion of solid foods and the source of nutrients for the queen and adult workers. It has been proposed that proteinase inhibitors might be a method for controlling undesirable insects; however, the use of non-specific inhibitors that inhibit a large number of ant proteolytic enzymes may cause a colony to relocate. Travis et al. (Acta Biochemica Polonica, 43, 411-417 (1996)) disclose in a review the existence of three proteolytic enzymes isolated from the 4th instar larvae of S. invicta. However, the purification steps are not disclosed in sufficient detail to allow purification of the proteolytic enzymes.
The present invention represents a potential advance in the control and/or eradication of fire ants by describing the purification and characterization of fire ant proteinases. The present invention provides isolated polypeptides, preferably isolated from an ant, particularly a S. invicta 4th instar larvae, having amidolytic activity for cleavage of a peptide bond present in a target polypeptide. In particularly preferred embodiments, four proteinases have been successfully isolated from the 4th instar larvae of S. invicta and characterized. Based on substrate specificity, they appear to represent two chymotrypsin-like and two elastase-like proteinases. These are referred to as Soli C1, Soli C2, Soli E1, and Soli E2, respectively, and have molecular weights of 25, 28, 23, and 24 kDa, respectively, based on SDS-PAGE. All enzymes are inhibited by diisopropyl fluorophosphate, a general serine class inhibitor. Each enzyme has been characterized as to pH optimum, pH stability, isoelectrofocusing, and susceptibility to inhibition by a broad range of natural and synthetic proteinase inhibitors. Such compounds may prove useful for the development of insecticides to control fire ant infestation.
In preferred embodiments, the polypeptide can have an elastase-like amidolytic activity, and can cleave a target polypeptide of MeOSuc-Ala-Ala-Pro-Val-pNA (SEQ ID NO:8), where the polypeptide and target polypeptide are in about 0.05 M Tris-HCl, about 100 mM NaCl at about pH 7.4 and about 25xc2x0 C. for about 10 minutes. In other preferred embodiments, the polypeptide can have a chymotrypsin-like amidolytic activity, and can cleave a target polypeptide of Suc-Ala-Ala-Pro-Phe-pNA (SEQ ID NO:7), where the polypeptide and target polypeptide are in about 0.05 M Tris-HCl, about 100 mM NaCl at about pH 7.4 and about 25xc2x0 C. for about 10 minutes. The polypeptide can include an amino terminal amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
The invention is also directed at an isolated polypeptide, an active analog or an active fragment thereof, where the polypeptide includes an amino acid sequence that has a percentage amino acid identity of greater than 39% with SEQ ID NO:2, or alternatively including the amino acid sequence of SEQ ID NO:2, an active analog or an active fragment thereof.
Another aspect of the invention is an isolated nucleic acid fragment encoding a polypeptide, where the nucleic acid fragment has a nucleotide sequence including nucleotides 519 to 1198 of SEQ ID NO:1, and a complement thereto. Alternatively, the nucleic acid fragment of the invention is a complement of the nucleic acid fragment that hybridizes to nucleotides 519 to 1198 of SEQ ID NO:1 under hybridization conditions of: a solution of 5 x Denhardt""s solution, 0.12 M phosphate buffer, pH 6.8 and 3 x SSC, at 47xc2x0 C. for about 12 hours, followed by two washes in a solution of 1 x SSC and 0.1% SDS for 20 minutes at 42xc2x0 C., and two washes in a solution containing 1 x SSC for 20 minutes at 42xc2x0 C., and a complement to the nucleic acid fragment.
The invention further provides an isolated polypeptide having chymotrypsin-like amidolytic activity for cleavage of a peptide bond present in a target polypeptide, where the polypeptide includes an amino terminal amino acid sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:26.
Alternatively, an isolated polypeptide having chymotrypsin-like amidolytic activity for cleavage of a peptide bond present in a target polypeptide is isolated from a Solenopsis invicta 4th instar larvae. An isolated polypeptide having chymotrypsin-like amidolytic activity isolated from a Solenopsis invicta 4th instar larvae can have a molecular weight of about 25 kDa or about 28 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An isolated polypeptide having chymotrypsin-like amidolytic activity isolated from a Solenopsis invicta 4th instar larvae can include an amino terminal amino acid sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:26.
The invention further provides an isolated polypeptide having elastase-like amidolytic activity for cleavage of a peptide bond present in a target polypeptide, where the polypeptide includes an amino terminal amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28.
Alternatively, an isolated polypeptide having elastase-like amidolytic activity for cleavage of a peptide bond present in a target polypeptide is isolated from a Solenopsis invicta 4th instar larvae. An isolated polypeptide having elastase-like amidolytic activity isolated from a Solenopsis invicta 4th instar larvae can have a molecular weight of about 23 kDa or about 24 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An isolated polypeptide having elastase-like amidolytic activity isolated from a Solenopsis invicta 4th instar larvae can include an amino terminal amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28.
An aspect of the invention includes methods of identifying a molecule that inhibits an amidolytic activity of a Solenopsis invicta 4th instar larvae polypeptide. The methods include incubating a polypeptide of the present invention with the molecule under conditions that promote amidolytic activity and determining if the amidolytic activity of the polypeptide of the present invention is inhibited (i.e., the level and/or rate of activity is at least partially decreased) relative to the amidolytic activity in the absence of the molecule. The polypeptide can be an isolated polypeptide having amidolytic activity for cleavage of a peptide bond present in a target polypeptide, where the polypeptide is isolated from an ant. Alternatively, the polypeptide can be an isolated polypeptide, an active analog or an active fragment thereof, the polypeptide including an amino acid sequence having a percentage amino acid identity of greater than 39% with SEQ ID NO:2. In another embodiment the polypeptide can be a polypeptide including an amino terminal amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.
Another aspect of the invention is a method of reducing the amidolytic activity of at least one polypeptide used in the digestion of solid foods by a Solenopsis invicta 4th instar larvae including contacting the at least one polypeptide with an inhibitor of the at least one polypeptide, wherein the at least one polypeptide comprises an amino terminal amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28. Preferably, the amidolytic activity of one polypeptide is inhibited.
The invention also includes a method of reducing the amount of solid food digested by a Solenopsis invicta 4th instar larvae including introducing an inhibitor to a Solenopsis invicta colony, where the inhibitor reduces the amidolytic activity of at least one polypeptide comprising an amino terminal amino acid sequence selected from at least one of the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide, whether synthetic or naturally occuring. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
xe2x80x9cProtease,xe2x80x9d xe2x80x9cproteinase,xe2x80x9d xe2x80x9cpeptidase,xe2x80x9d and xe2x80x9cproteolytic enzymexe2x80x9d all refer to a polypeptide that catalyzes the hydrolysis of peptide bonds in a polypeptide. A xe2x80x9cpeptide bondxe2x80x9d or xe2x80x9camide bondxe2x80x9d is a covalent bond between the alpha-amino group of one amino acid and the alpha-carboxyl group of another amino acid. xe2x80x9cPeptidase inhibitor,xe2x80x9d xe2x80x9cproteinase inhibitor,xe2x80x9d xe2x80x9cprotease inhibitor,xe2x80x9d and xe2x80x9cinhibitorxe2x80x9d all refer to molecules that inhibit a protease that catalyzes the hydrolysis of peptide bonds in a polypeptide.
As used herein, the term xe2x80x9cisolatedxe2x80x9d means that a nucleic acid fragment or polypeptide is either removed from its natural environment or synthetically derived. Preferably, the nucleic acid fragment or polypeptide is purified, i.e., essentially free from any other nucleic acid fragments or polypeptides and associated cellular products or other impurities. xe2x80x9cAmidolytic activityxe2x80x9d and xe2x80x9cprotease activityxe2x80x9d refer to the ability of a polypeptide to catalyze the hydrolysis of at least one peptide bond in a polypeptide. The term xe2x80x9ccleavagexe2x80x9d can also be used to refer to the hydrolysis of a peptide bond in a polypeptide. A polypeptide having xe2x80x9cchymotrypsin-likexe2x80x9d amidolytic activity is a polypeptide having amidolytic activity similar to chymotrypsin in that it cleaves a peptide bond in substrates having a P1 residue of leucine or phenylalanine. A polypeptide having xe2x80x9celastase-likexe2x80x9d amidolytic activity is a polypeptide having amidolytic activity similar to pancreatic elastase in that it cleaves a peptide bond in substrates having a P1 residue of alanine or valine. P1 refers to the amino acid residue immediately upstream of the cleaved peptide bond.
A xe2x80x9ctarget polypeptidexe2x80x9d is a polypeptide that is the potential substrate for the amidolytic activity of a protease.
An xe2x80x9cactive analogxe2x80x9d or xe2x80x9cactive fragmentxe2x80x9d of a polypeptide of the invention is one that has amidolytic activity by hydrolysis of a peptide bond present in the target polypeptide as described herein. Active analogs and active fragments are described in greater detail herein.
xe2x80x9cNucleic acid fragmentxe2x80x9d as used herein refers to a linear polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A nucleic acid fragment may include both coding and non-coding regions that can be obtained directly from a natural source (e.g., an ant), or can be prepared with the aid of recombinant or synthetic techniques. A nucleic acid molecule may be equivalent to this nucleic acid fragment or a nucleic acid molecule can include this fragment in addition to one or more other nucleotides or polynucleotides. For example, a nucleic acid molecule of the invention can be a vector, such as an expression or cloning vector.
xe2x80x9cPercentage amino acid identityxe2x80x9d and xe2x80x9cpercentage nucleic acid identityxe2x80x9d refer to a comparison of the amino acids of two polypeptides or a comparison of the nucleotides of two nucleic acid sequences, respectively, as described herein.
xe2x80x9cAntxe2x80x9d as used herein refers to social insects of the family Formicidae, typically having wings only in the males and fertile females, and living in colonies that have a complex social order. xe2x80x9cAntxe2x80x9d includes all developmental stages; egg, 1st instar larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, prepupae, pupae, and adult.
Unless otherwise specified, the indefinite article xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d means one or more.